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  • C12N5/0602—Vertebrate cells
  • C12N5/0603—Embryonic cells ; Embryoid bodies
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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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  • C12N5/069—Vascular Endothelial cells
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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/069—Vascular Endothelial cells
  • C12N5/0691—Vascular smooth muscle cells; 3D culture thereof, e.g. models of blood vessels
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  • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

Definitions

• the present invention relates to a method for isolating four cell types from the umbilical cord, and more particularly from the umbilical vein, in good conditions without having to execute separate methods for each type of cell.
• the method is of particular importance for obtaining a variety of different cells in good condition from only one tissue.
• stem cells are capable of self-regeneration but may also divide into progenitor cells that are no longer pluripotent nor capable of self- regeneration. These progenitor cells divide repeatedly to form more mature cells, which eventually become terminally differentiated to form various mature cells. Thus the large number of mature cells is derived from a small reservoir of stem cells by a process of proliferation and differentiation.
• tissue-engineered blood vessels should provide mechanically stable vessels built only from autologous tissue, therefore generating no immune responses.
• Tissue engineering has been used successfully in the past to build less complex structures such as skin, but has had only relative success with other three-dimensional tissues and organs such as tissue-engineered blood vessels.
• Common problems associated with three- dimensional engineered tissues include the complexity of reconstruction, the lack of structural integrity and mechanical strength, and the need for biologically active tissues.
• tissue-engineered blood vessels This is a particular problem for tissue-engineered blood vessels, since these vessels will be subjected to significant mechanical loads both from blood pressure (which may be abnormally high in patients with heart disease), as well as from the relative motion between the anchoring sites of the vessel.
• the tissue-engineered blood vessels must demonstrate sufficient stability and tear resistance to allow surgical handling and implantation, and require a biologically active endothelial layer.
• An important cause of these problems seems to be associated with the sources of the cells used to prepare engineered blood vessels or other replacement tissues. In fact, most methods available at the moment allow the isolation of a maximum of two cell types from a single tissue, which should be preferentially autologous.
• these cells are generally mature and well differentiated, which constitutes a limiting factor when a certain level of flexibility and adaptability is necessary in constructing an engineered replacement tissue.
• Stem cells mature into progenitor cells and then become lineage committed, that is, incapable of maturing into all of the different lineages which the stem cell is capable of producing.
• Highly purified populations of stem cells currently find use in the long-term repopulation of particular body systems.
• Purified progenitor cells of individual lineages would find use only in transiently repopulating or augmenting the various lineages. As progenitors are not believed to be self-regenerating, the repopulation or augmentation would be limited, for example, to short-term tissue-specific reconstitution.
• the field of tissue engineering uses living cultured human or animal cells from various sources to reconstruct functional tissues and organs for experimental and therapeutic purposes.
• Medical applications of umbilical haematopoietic cord stem cells are becoming well known both in and out of the medical community.
• cord haematopoietic stem cells to date (or who will benefit from cord stem cell uses in the future)
• different cord stem cell preparations are presently under investigation.
• the use of the pluripotentiality of lineage-committed progenitor cells circumvents many of the problems that would arise from the transfer of mature cells. However, such progenitor cells may have to be separated through carrying out parallel methods upon different sources of living tissues.
• a method for the simultaneous isolation of four cell types from one umbilical cord sample comprising the steps of: a) isolating endothelial cells by perfusing the vein of an umbilical cord biopsy with a solution comprising thermolysin for a period of time to allow detachment of said endothelial cells from said vein; b) treating said umbilical cord biopsy of step a) with a solution of trypsin-EDTA for a time sufficient to allow detachment of epithelial cells; c) mechanically removing smooth muscle cells and fibroblast-like cells from said umbilical cord biopsy followed by a culture period of sufficient time to allow separation and isolation of smooth muscle and fibroblast-like cells from laminae; and d) harvesting the isolated cells of steps a) to c).
• the invention also provides for the use of the above-mentioned four cell types for the preparation of a replacement or engineered tissue or graft
• the invention additionally relates to a biological composition comprising these four cell types.
• Fig. 1 illustrates the typical aspects of the four cell types in monolayer culture, in phase contrast photographs;
• Fig. 2 illustrates sections of the umbilical cord before and after the extraction of endothelial cells, and endothelial cells in culture immunolabelled for van Willebrand factor ;
• Fig. 3 illustrates sections of the umbilical cord before and after the extraction of smooth muscle cells, and smooth muscle cells in culture immunolabelled for ⁇ -smooth muscle actin ; Fig.
• Fig. 4 illustrates sections of the umbilical cord before and after the extraction of fibroblasts, and fibroblasts in culture immunolabelled for vimentin; and Fig. 5 illustrates sections of the umbilical cord before and after the extraction of epithelial cells, and epithelial cells in culture immunolabelled for desmoplakin.
• the advantage of the present invention over the prior art will be recognized as providing a method allowing the preparation of four umbilical cell types in which after each step, all the desired types of cells are kept alive and in condition to be harvested for further uses.
• No equivalent techniques in the art are known to allow a skilled person to isolate four cell types from a single tissue biopsy without significant cell mortality and tissue destruction.
• the inventors have developed a method for the extraction of four distinct cell types from a single umbilical cord biopsy or section. This permits the conservation of multiple cell types from a unique source, or from only one person, for present or future therapeutic applications, and this also permits research using tissue models fabricated with cells of the same lineage.
• autologous cells are harvested from the patient's own body to eliminate the risks of disease transmission and tissue rejection.
• All umbilical cord biopsies contain endothelial cells, epithelial cells, smooth muscle cells, and fibroblasts. Therefore, almost any biopsy procedure or tissue harvest will provide a suitable starting point for the four of them.
• the umbilical cord is of particular interest as a source of cell lines because of its foetal condition, and because its obtention is simple and non- invasive.
• the present invention is directed to a method for the extraction of four cell types: epithelial cells, fibroblasts, smooth muscle cells and endothelial cells, as pure cultures from a single human umbilical cord.
• the human umbilical cord is a foetal structure that carries blood from the foetal circulation to the placenta for oxygenation via the two umbilical arteries, and returns the oxygenated blood to the developing child by way of the umbilical vein.
• the blood vessels are lined with endothelial cells, which are in turn surrounded by a substantial layer of smooth muscle cells (SMC), thicker around the arteries than around the vein.
• SMC smooth muscle cells
• the bundle of blood vessels is enclosed in turn by a thick layer of extracellular matrix sparsely populated by fibroblasts and known as Wharton's jelly.
• a thin layer of epithelial cells covers the outer surface of the umbilical cord.
• the umbilical cord epithelium is formed of a thin layer of epithelial cells resting on a basement membrane, and is the only part of the umbilical cord to be in contact with the surrounding amniotic fluid, thus acting as a barrier between the internal tissues and the outer liquid. It is often only a single cell layer thick, but regions of up to five layers of thickness do occur.
• the epithelium of the cord is contiguous both with the amniotic epithelium, from which it is derived in the early stages of development, as well as with the early embryonic periderm and the later foetal epithelium.
• the morphology of the umbilical cord epithelium has been described as being closely related to the early foetal epidermis before its keratinisation.
• This process does not occur normally in the umbilical cord epithelium except in the region adjacent to the foetus, the rest of the epithelium remaining as a simple squamous epithelium.
• the epithelial cells are tightly joined to each other by numerous desmosomes, and to the underlying basement membrane by hemidesmosomes.
• the dense cytoskeleton contains keratin fibres of many types, the presence of which is typical of epithelia. Few studies have been done to further characterize these cells.
• the connective tissue of the umbilical cord commonly called Wharton's jelly, consists of fibroblasts dispersed within a loose mucous connective tissue, composed mainly of a network of collagen fibres and a ground substance of glycosaminoglycans, mostly hyaluronic acid, along with an independent fibrillar network of glycoprotein microfibrils. This tissue serves to protect the umbilical blood vessels from compression and torsion.
• the fibroblasts themselves are somewhat unusual as they combine characteristics of both typical fibroblastic cells (abundant Golgi apparati, collagen secretion granules, mitochondria and rough endoplasmic reticulae) and of smooth muscle cells (deeply indented nuclei, pinocytotic vesicles and fibronexus junctions). Their content of ⁇ -smooth muscle actin gives them a certain contractility. Desmin and non-muscle myosin have also been identified as intracellular components. There is disagreement on the exact nature and origin of these cells because of their diverse characteristics, and further analysis will be necessary to shed light on their exact role. Smooth muscle cells form the ring-shaped media that surround the arteries and vein of the umbilical cord.
• smooth muscle cells Their main function is contraction, but these cells are also capable of many other functions when required, including production of extracellular matrix proteins and increased cell proliferation.
• Typical components of the smooth muscle cell include smooth muscle actin, myosin, calponin, caldesmon, vinculin, tropomyosin, vimentin and desmin. Many and varied ion channels and membrane receptors allow sensitive regulation of the contractile behaviour of these cells. Smooth muscle cells derived from the umbilical cord have been extensively used in the study of smooth muscle cells, their metabolism and their characteristics, as well as in tissue reconstructions.
• the umbilical cord vein and arteries are lined by endothelial cells, which have many diverse functions in the body, including the maintenance of a non- thrombogenic intimal surface, the regulation of coagulation and fibrinolysis, immunological functions, the regulation of perfusion and permeability across the vessel walls, and the release of and response to chemical agents.
• Endothelial cells harvested from umbilical cord blood vessels and other sources have been used for numerous experimental purposes for many years, especially for studies on angiogenesis and its control by pharmacological manipulation, and have been well characterized. They are known by their typical cobblestone shape in monolayer culture, and their ability to form tubules when plated in three-dimensional gels and collagen sponges.
• Universal cellular markers for endothelial cells include the adhesion molecule CD-31 and the von Willebrand factor. These cell types can be isolated from the umbilical cord by a variety of techniques, purified, and expanded in culture.
• the object of the present invention is to provide a method to isolate pure cell lines of four different cell types from a single umbilical cord. The cell lines can then be utilised in the reconstruction of multilineage tissues, including but not limited to vascular structures. These reconstructions are of interest and value not only for the study of the interactions between cells and cell types but also in a long-term therapeutic view, wherein the cells from a person's umbilical cord could be conserved and ultimately serve in the replacement of diseased or damaged tissues.
• Endothelial cells, epithelial cells, fibroblasts and smooth muscle cells can be isolated from the biopsy by techniques described herein.
• the invention essentially consists of enzymatic digestion i specific media and manual dissection of the umbilical cord tissue to separate the cell-containing tissues. Fibroblasts and smooth muscle cells can be harvested from tissue explants of the biopsy by cell outgrowth or by enzymatically digesting the explants and plating the digested tissue. After the four umbilical cell types have been isolated, they must be cultured and grown into engineered tissues with sufficient mechanical stability and strength to be detached and organized into a three-dimensional living structure.
• Mature engineered living structures of isolated cells can include, but are not limited to, their own extracellular matrix proteins. Additional cells can be added to the four cell types at any stage of tissue formation. These cells can include additional human or animal cells or transfected or otherwise genetically modified cells.
• One particular embodiment of the present invention is to provide a composition of four cell types isolated from the same autologous source and having a high progenitor potential.
• An embodiment of the invention is directed to the use of neonatal progenitor cells for living or biological tissue reconstitution. There are several reasons for preferring the use of such neonatal cells to that of conventional mature cells. First, no donor is required because the cells can be obtained from neonatal umbilical cords that would otherwise be discarded.
• the present method allows the provision of vascular cells for neovascularisation, and of fibroblastic cells for the reconstruction of mesenchymal structures such as ligament or cartilages.
• fibroblastic cells have successfully been used in the allogeneic reconstruction of dermal tissue, and thus such allogeneic applications could equally be envisaged for the fibroblasts and other cell types of the umbilical cord.
• neonatal cells for tissue reconstitution and engineering as provided by the present invention.
• the neonatal umbilical cord is a preferred source of cells for tissue reconstitution and engineering, since it is much less prone to microbial and viral contamination, known or unknown, latent or otherwise, that may be encountered in later life, other than those transmitted from the mother or during labor and delivery.
• the known stem cells may possibly share with other cells the limitation in the total number of cell divisions that they may undergo before senescence, it is proper to assume that the neonatal umbilical progenitor cells have a self-renewal and reconstitutional capacity that is at least as great, and perhaps even greater, than that of cells obtained at any later time in life.
• the present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.
• the umbilical cord was gently cleansed of blood and debris on the outer surface with a moist sterile gauze, and inspected for clamp marks, cuts and other analomies which might have damaged the internal structure of the cord and thus cause a mixing of the cell types extracted.
• An undamaged section of the umbilical cord preferably greater than 15 cm in length, was used for the extraction of the endothelial cells.
• the umbilical vein on one end of the section was cannulated using a small plastic adapter, a plastic tube attached to a stopcock capable of receiving a syringe, and a circular clamp, and the vein was rinsed 3 times with 10 mL of cold sterile Hepes IX.
• the cord was removed from the container and very gently massaged in order to dislodge all the endothelial cells possible.
• the vein was then rinsed with 30 mL warm Hepes and the perfusion liquid collected in a centrifugation tube partially filled with Ml 99 complete medium.
• the cells were then pelleted by centrifugation, resuspended and plated in gelatinated culture flasks with the same medium. Isolation of epithelial cells
• the section of the umbilical cord used for the extraction of endothelial cells was subsequently used for the extraction of the epithelial cells.
• the greatest portion possible of the cord was placed in a sterile container with slots cut in opposing sides to hold the ends of the cord out of the enzymatic solution.
• the container was then filled as full as possible with approximately 40 mL of trypsin 0.25% EDTA, sealed and placed at 37°C with gentle agitation for 5 min.
• the enzymatic solution was then discarded and replaced with a fresh aliquot. Successive incubation times of 15, 15, and 30 min. were then undertaken. After each incubation period serum was added as a trypsin inhibitor to the collected solution of trypsin, and the mixture was centrifuged.
• the pelleted cells from each fraction were resuspended in complete DME-Ham's 10% CS and pooled in order to perform a count of the cells obtained.
• the cells were seeded in culture flasks with a feeder layer of murine Swiss 3T3 irradiated fibroblasts (S3T3) (20 000 cells/cm 2 ) at a density of 80 000 cells/cm 2 in DME-Ham's complete medium.
• Culture Media Endothelial cells Ml 99 (Sigma # 5017) reconstituted with apyrogenic water and 2.2 g/L sodium bicarbonate, 20 % calf serum (Hyclone), 20 mg/mL endothelial cell growth factor (Sigma E-2759), 2.28 mM glutamine (GLNS), 0.40 U/rnL heparin, 100 UI/mL penicillin G, 25 mg/mL gentamycin sulphate.
• Smooth muscle cells Dulbecco-Vogt modified Eagle's medium and Ham's F12 (3:1 mixture), 10 % foetal calf serum (Biomedia), 100 UI/mL penicillin G, 25 mg/mL gentamycin sulphate.
• Fibroblasts Dulbecco-Vogt modified Eagle's medium, 10 % foetal calf serum (Hyclone) 100 UI/mL penicillin G, 25 mg/mL gentamicin sulphate.
• Epithelial cells Dulbecco-Vogt modified Eagle's medium and Ham's F12
• Cells in Culture All four cell types could be established and maintained in culture using the aforementioned combination of extraction techniques. Each cell type has its characteristic phenotype and proliferation profile, as well as its particular requirements insofar as culture conditions, h Figure 1, the typical aspects of the different cell types in monolayer culture are illustrated; in a) endothelial cells, in b) smooth muscle cells, in c) fibroblasts, and in d) a proliferative colony of epithelial cells surrounded by feeder S3T3 cells. All photographs were taken at 10X magnification under phase contrast. Problems encountered with the extractions were not directly related to the extraction technique itself, but resulted from a variation of the properties of tissues and cells between different individuals.
• FIG. 2a shows a section of umbilical cord vein with the layer of endothelial cells overlying the media (smooth muscle cells), at 40X magnification. After the extraction of the endothelial cells, an intact media remains ( Figure 2b, 20X magnification).
• Figure 3a shows a section of umbilical cord vein with the layers of smooth muscle cells overlying the connective tissue called Wharton's jelly, 40X magnification.
• FIG. 4a a section of umbilical cord shows' the connective tissue called Wharton's jelly surrounding part of the umbilical cord vein, 10X magnification. After extraction of the fibroblasts, Figure 4b shows no ingression into the media surrounding the vein (4X magnification).
• Figure 5a a section of umbilical cord shows the epithelium overlying the connective tissue called Wharton's jelly, at 40X magnification.
• Figure 5b shows no break in the basement membrane underlying the epithelial cells and no ingression into the connective tissue of Wharton's jelly (40X magnification).
• the cells in culture displayed homogenous populations of proliferative cells with characteristic phenotypes. No cultures of mixed cell types were noted in any of the extractions studied (see Figure 1).
• the identity of the cell populations was verified by immunofluorescent marking of cell type-specific protein markers in cells grown on slides and fixed in acetone.
• Figure 2c shows the presence of von Willebrand factor in the endothelial cells
• Figure 3c illustrates the marking of smooth muscle ⁇ -actin in smooth muscle cells
• Figure 4c demonstrates the presence of vimentin, a structural protein common to fibroblastic cells, in the fibroblasts of Wharton's jelly (all at 40X magnification)
• Figure 5c shows the marking of desmoplakin, a protein of adhesion particular to epithelial cells, in cultured epithelial cells (60X magnification).
• Characterisation of Cell Types The endothelial cells and smooth muscle cells of the human umbilical cord have been extensively studied to date by various research groups, and details of their known characteristics can be found in many publications. Epithelial cells and the fibroblasts from Wharton's jelly, however, have received much less attention and we present here a summary of the immunofluorescent markings carried out on these two cell types both in vivo and in vitro.

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